Various publications are referred to in parentheses throughout this application. Full citations for these references may be found at the end of the specification immediately preceding the claims. The disclosures of these publications are hereby incorporated by reference in their entireties into the subject application to more fully describe the art to which the subject application pertains.
One out of three cancers diagnosed among U.S. women is due to breast cancer; 212,920 new invasive breast cancer cases and an additional 61,980 in situ breast cancer cases are expected to be diagnosed in the U.S. in 2006. Around 40,970 women are expected to die from breast cancer in 2006 in the U.S. alone (American Cancer Society, Breast Cancer Facts and Figures 2006). The metastasis of 10-15% of patients with breast cancer is aggressive and can take between 3-10 years to be manifested after the initial diagnosis. Currently, the prognosis in 70% of patients cannot be accurately determined resulting in the unnecessary treatment of many patients who will not benefit and may be injured by radiation and chemotherapy. The availability of an antibody and associated polymerase chain reaction (PCR) primer pair that uniquely and specifically identifies metastatic disease will allow for accurate prediction of disease course and allow appropriate treatment.
Invasion of tumor cells into surrounding tissue and intravasation into blood and lymphatic vessels is implicated in the progression of metastatic breast cancer. This multi-step process involves a number of phenotypic changes which occur sequentially and give rise to a hyper-invasive cell (Condeelis et al., 2005). In an effort to identify these individual events and to understand the molecular events underlying these phenotypic changes, animal models have been developed as well as a chemotaxis assay that isolates the in vivo invasive cells from the average primary tumor cells (APTC) (Wyckoff et al., 2000). Chemotaxis based isolation of the invasive cells and subsequent gene expression analysis have resulted in the identification of an invasion specific gene expression signature in invasive cells (Wang et al., 2004). In these studies a number of genes have been identified which need to be co-ordinately up-regulated in the invasive cells in order for invasion to lead to metastasis (Wang et al., 2006).
One of the key genes of the invasion signature is that coding for the cytoskeletal protein Mena. Mena is a member of the Ena/VASP family of proteins. These proteins are regulatory molecules which control cell movement, motility and shape in a number of cell types and organisms. They are proposed to function by preventing the actin filaments from being capped by capping proteins at their barbed ends (Barzik et al., 2005). The anti-capping activity of Mena has been proposed to amplify the barbed end output of the cofilin and Arp2/3 complex pathways, which is sufficient to increase metastatic potential in mammary tumors (Wang et al., 2006). Ena/VASP proteins are also constituents of the adherence junctions necessary to seal membranes in the epithelial sheet and control actin organization on cadherin adhesion contact (Scott et al., 2006). This process is frequently perturbed in cancer. Ena/VASP proteins contain specific domains including the N-terminal EVH1 domain, which plays an essential role in intracellular protein localization by interacting with proline-rich motifs found in proteins such like zyxin and vinculin (Prehoda et al., 1999). The proline-rich domain in the center is known to mediate interaction with proteins having the SH3 and WW domains and also with the actin monomer binding protein profilin (Gertler et al., 1996). The C-terminal domain of Mena contains an EVH2 domain that is involved in tetramerization of the protein and also binding to G- and F-actin (Kuhnel et al., 2004). The interaction of the EVH2 domain with the growing ends of the actin filaments is essential for targeting the Ena/VASP to lamellipodia and filopodia (Loureiro et al., 2002). Mena is upregulated in mouse and rat invasive breast cancer cells (Wang et al., 2004) and overexpressed in human breast cancer tissues (Di Modugno et al., 2004). Both mouse and human Mena homologs have been cloned and sequenced, and a number of splice variants have been identified (Gertler et al., 1996; Urbanelli et al., 2006).
Recently it has been shown that splice variants can work very efficiently as cancer biomarkers (Brinkman 2004; Venables 2006). However, there remains a need to identify splice variants that are upregulated specifically in metastatic cancer cells, such as metastatic breast cancer cells.